IV BCG Vaccination and Aerosol BCG Revaccination Induce Mycobacteria-Responsive γδ T Cells Associated with Protective Efficacy against M. tb Challenge.

Intravenously (IV) delivered BCG provides superior tuberculosis (TB) protection compared with the intradermal (ID) route in non-human primates (NHPs). We examined how γδ T cell responses changed in vivo after IV BCG vaccination of NHPs, and whether these correlated with protection against aerosol M. tuberculosis challenge. In the circulation, Vδ2 T cell populations expanded after IV BCG vaccination, from a median of 1.5% (range: 0.8-2.3) of the CD3+ population at baseline, to 5.3% (range: 1.4-29.5) 4 weeks after M. tb, and were associated with TB protection. This protection was related to effector and central memory profiles; homing markers; and production of IFN-γ, TNF-α and granulysin. In comparison, Vδ2 cells did not expand after ID BCG, but underwent phenotypic and functional changes. When Vδ2 responses in bronchoalveolar lavage (BAL) samples were compared between routes, IV BCG vaccination resulted in highly functional mucosal Vδ2 cells, whereas ID BCG did not. We sought to explore whether an aerosol BCG boost following ID BCG vaccination could induce a γδ profile comparable to that induced with IV BCG. We found evidence that the aerosol BCG boost induced significant changes in the Vδ2 phenotype and function in cells isolated from the BAL. These results indicate that Vδ2 population frequency, activation and function are characteristic features of responses induced with IV BCG, and the translation of responses from the circulation to the site of infection could be a limiting factor in the response induced following ID BCG. An aerosol boost was able to localise activated Vδ2 populations at the mucosal surfaces of the lung. This vaccine strategy warrants further investigation to boost the waning human ID BCG response.

Killer to cure: Expression and production costs calculation of tobacco plant-made cancer-immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) have achieved huge clinical success. However, many still have limited response rates, and are prohibitively costly. There is a need for effective and affordable ICIs, as well as local manufacturing capacity to improve accessibility, especially to low-to-middle income countries (LMICs). Here, we have successfully expressed three key ICIs (anti-PD-1 Nivolumab, anti-NKG2A Monalizumab, and anti-LAG-3 Relatimab) transiently in Nicotiana benthamiana and Nicotiana tabacum plants. The ICIs were expressed with a combination of different Fc regions and glycosylation profiles. They were characterized in terms of protein accumulation levels, target cell binding, binding to human neonatal Fc receptors (hFcRn), human complement component C1q (hC1q) and various Fcγ receptors, as well as protein recovery during purification at 100 mg- and kg-scale. It was found that all ICIs bound to the expected target cells. Furthermore, the recovery during purification, as well as Fcγ receptor binding, can be altered depending on the Fc region used and the glycosylation profiles. This opens the possibility of using these two parameters to fine-tune the ICIs for desired effector functions. A scenario-based production cost model was also generated based on two production scenarios in hypothetical high- and low-income countries. We have shown that the product accumulation and recovery of plant production platforms were as competitive as mammalian cell-based platforms. This highlights the potential of plants to deliver ICIs that are more affordable and accessible to a widespread market, including LMICs.

Releasing the restraints of Vγ9Vδ2 T-cells in cancer immunotherapy.

Objectives: Vγ9Vδ2 T-cells are a subset of T-cells with a crucial role in immunosurveillance which can be activated and expanded by multiple means to stimulate effector responses. Little is known about the expression of checkpoint molecules on this cell population and whether the ligation of these molecules can regulate their activity. The aim of this study was to assess the expression of both activatory and inhibitory receptors on Vγ9Vδ2 T-cells to assess potential avenues of regulation to target with immunotherapy. Methods: Expression of various activatory and inhibitory receptors was assessed on Vγ9Vδ2 T-cells by flow cytometry following activation and expansion using zoledronic acid (ZA) and Bacillus Calmette-Guérin (BCG). Expression of these markers and production of effector molecules was also examined following co-culture with various tumour cell targets. The effect of immune checkpoint blockade on Vγ9Vδ2 T-cells was also explored. Results: Vγ9Vδ2 T-cells expressed high levels of activatory markers both at baseline and following stimulation. Vγ9Vδ2 T-cells expressed variable levels of inhibitory checkpoint receptors with many being upregulated following stimulation. Expression of these markers is further modulated upon co-culture with tumour cells with changes reflecting activation and effector functions. Despite their high expression of inhibitory receptors when cultured with tumour cells expressing cognate ligands there was no effect on Vδ2+ T-cell cytotoxic capacity or cytokine production with immune checkpoint blockade. Conclusions: Our work suggests the expression of checkpoint receptors present on Vγ9Vδ2 T-cells which may provide a mechanism with the potential to be utilised by tumour cells to subvert Vγ9Vδ2 T-cell cytotoxicity. This work suggests important candidates for blockade by ICI therapy in order to increase the successful use of Vγ9Vδ2 T-cells in immunotherapy.

Cheap and Commonplace: Making the Case for BCG and γδ T Cells in COVID-19.

Antigen-specific vaccines developed for the COVID-19 pandemic demonstrate a remarkable achievement and are currently being used in high income countries with much success. However, new SARS-CoV-2 variants are threatening this success via mutations that lessen the efficacy of antigen-specific antibodies. One simple approach to assisting with this issue is focusing on strategies that build on the non-specific protection afforded by the innate immune response. The BCG vaccine has been shown to provide broad protection beyond tuberculosis disease, including against respiratory viruses, and ongoing studies are investigating its efficacy as a tool against SARS-CoV-2. Gamma delta (γδ) T cells, particularly the Vδ2 subtype, undergo rapid expansion after BCG vaccination due to MHC-independent mechanisms. Consequently, γδ T cells can produce diverse defenses against virally infected cells, including direct cytotoxicity, death receptor ligands, and pro-inflammatory cytokines. They can also assist in stimulating the adaptive immune system. BCG is affordable, commonplace and non-specific, and therefore could be a useful tool to initiate innate protection against new SARS-CoV-2 variants. However, considerations must also be made to BCG vaccine supply and the prioritization of countries where it is most needed to combat tuberculosis first and foremost.

How to Train Your Dragon: Harnessing Gamma Delta T Cells Antiviral Functions and Trained Immunity in a Pandemic Era.

The emergence of viruses with pandemic potential such as the SARS-CoV-2 coronavirus causing COVID-19 poses a global health challenge. There is remarkable progress in vaccine technology in response to this threat, but their design often overlooks the innate arm of immunity. Gamma Delta (γδ) T cells are a subset of T cells with unique features that gives them a key role in the innate immune response to a variety of homeostatic alterations, from cancer to microbial infections. In the context of viral infection, a growing body of evidence shows that γδ T cells are particularly equipped for early virus detection, which triggers their subsequent activation, expansion and the fast deployment of antiviral functions such as direct cytotoxic pathways, secretion of cytokines, recruitment and activation of other immune cells and mobilization of a trained immunity memory program. As such, γδ T cells represent an attractive target to stimulate for a rapid and effective resolution of viral infections. Here, we review the known aspects of γδ T cells that make them crucial component of the immune response to viruses, and the ways that their antiviral potential can be harnessed to prevent or treat viral infection.

Influence of Aerosol Delivered BCG Vaccination on Immunological and Disease Parameters Following SARS-CoV-2 Challenge in Rhesus Macaques.

The tuberculosis vaccine, Bacille Calmette-Guerin (BCG), also affords protection against non-tuberculous diseases attributable to heterologous immune mechanisms such as trained innate immunity, activation of non-conventional T-cells, and cross-reactive adaptive immunity. Aerosol vaccine delivery can target immune responses toward the primary site of infection for a respiratory pathogen. Therefore, we hypothesised that aerosol delivery of BCG would enhance cross-protective action against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and be a deployable intervention against coronavirus disease 2019 (COVID-19). Immune parameters were monitored in vaccinated and unvaccinated rhesus macaques for 28 days following aerosol BCG vaccination. High-dose SARS-CoV-2 challenge was applied by intranasal and intrabronchial instillation and animals culled 6-8 days later for assessment of viral, disease, and immunological parameters. Mycobacteria-specific cell-mediated immune responses were detected following aerosol BCG vaccination, but SARS-CoV-2-specific cellular- and antibody-mediated immunity was only measured following challenge. Early secretion of cytokine and chemokine markers associated with the innate cellular and adaptive antiviral immune response was detected following SARS-CoV-2 challenge in vaccinated animals, at concentrations that exceeded titres measured in unvaccinated macaques. Classical CD14+ monocytes and Vδ2 γδ T-cells quantified by whole-blood immunophenotyping increased rapidly in vaccinated animals following SARS-CoV-2 challenge, indicating a priming of innate immune cells and non-conventional T-cell populations. However, viral RNA quantified in nasal and pharyngeal swabs, bronchoalveolar lavage (BAL), and tissue samples collected at necropsy was equivalent in vaccinated and unvaccinated animals, and in-life CT imaging and histopathology scoring applied to pulmonary tissue sections indicated that the disease induced by SARS-CoV-2 challenge was comparable between vaccinated and unvaccinated groups. Hence, aerosol BCG vaccination did not induce, or enhance the induction of, SARS-CoV-2 cross-reactive adaptive cellular or humoral immunity, although an influence of BCG vaccination on the subsequent immune response to SARS-CoV-2 challenge was apparent in immune signatures indicative of trained innate immune mechanisms and primed unconventional T-cell populations. Nevertheless, aerosol BCG vaccination did not enhance the initial clearance of virus, nor reduce the occurrence of early disease pathology after high dose SARS-CoV-2 challenge. However, the heterologous immune mechanisms primed by BCG vaccination could contribute to the moderation of COVID-19 disease severity in more susceptible species following natural infection.

The cytotoxic molecule granulysin is capable of inducing either chemotaxis or fugetaxis in dendritic cells depending on maturation: a role for Vδ2+ γδ T cells in the modulation of immune response to tumour?

Release of granulysin by γδ T cells contributes to tumour cell killing. A cytolytic 9000 MW isoform of granulysin kills tumour cells directly, whereas a 15 000 MW precursor has been hypothesized to cause both the maturation and migration of dendritic cell (DC) populations. Recruiting DC to a tumour is beneficial as these cells initiate adaptive immune responses, which contribute to the eradication of malignancies. In this study, Vδ2+ γδ T cells were activated by stimulation of peripheral blood mononuclear cells with zoledronic acid or Bacillus Calmette-Guérin (BCG), or were isolated and cultured with tumour targets. Although a large proportion of resting Vδ2+ γδ T cells expressed 15 000 MW granulysin, 9000 MW granulysin expression was induced only after stimulation with BCG. Increased levels of activation and granulysin secretion were also observed when Vδ2+ γδ T cells were cultured with the human B-cell lymphoma line Daudi. High concentrations of recombinant 15 000 MW granulysin caused migration and maturation of immature DC, and also initiated fugetaxis in mature DC. Conversely, low concentrations of recombinant 15 000 MW granulysin resulted in migration of mature DC, but not immature DC. Our data therefore support the hypothesis that Vδ2+ γδ T cells can release granulysin, which may modulate recruitment of DC, initiating adaptive immune responses.

Serum Apolipoprotein E and Other Inflammatory Markers Can Identify Non-Responding Patients to a Dendritic Cell Vaccine.

BACKGROUND: Despite the majority of patients do not gain any benefit from dendritic cells (DC) vaccines, this approach has occasionally given rise to dramatic responses in melanoma. Biomarkers are crucial to identify which patients are more likely to respond. We looked for correlations between pre- or post- vaccination biomarkers and clinical outcomes to DC therapy in a cohort of patients with stage IV melanoma receiving a vaccine with autologous ex-vivo expanded DCs pulsed with allogeneic tumor cell lysate. METHODS: Serial serum samples were collected at baseline, week 4 and 12 and they were analyzed for a panel of different inflammatory markers using cytometric bead array technology and ELISA. RESULTS: Twenty-one patients were evaluable for response. Patients were separated into responders and non-responders based on clinical benefit. Responders were defined as patients who achieved a complete response, partial response or stable disease the latter lasting for at least 6 months. Responders (N = 9) showed a significantly longer Progression-free Survival (PFS; HR 0.23; 95% CI 0.08-062; P < .001) and Overall Survival (OS; HR 0.22; 95% CI 0.08-0.59; P < .001). The clinical non-responder phenotype correlated with an elevated pre-vaccination level of cytokines associated with inflammation compared to clinical responders (Apolipoprotein C111; IL-12 p40; MiP1α; Stem Cell Factor and TNFα). Apolipoprotein E (ApoE) was also significantly elevated in the pre-vaccine sera of the clinically non-responding group and in addition it was found to correlate with outcomes. Patients with increased levels of ApoE had a significantly shorter PFS (HR 3.02; 95% CI 1.09-8.35; P = .015) and OS (HR 2.40; 95% CI 0.9-6.3; P = .034). CONCLUSION: Our findings support the notion that treating the inflammatory background may have an impact on clinical outcome for patients receiving immunotherapy. A larger study is needed to confirm the significance of ApoE as a predictive biomarker for response to DC vaccines.

Zoledronic acid renders human M1 and M2 macrophages susceptible to Vδ2+ γδ T cell cytotoxicity in a perforin-dependent manner.

Vδ2+ T cells are a subpopulation of γδ T cells in humans that are cytotoxic towards cells which accumulate isopentenyl pyrophosphate. The nitrogen-containing bisphosphonate, zoledronic acid (ZA), can induce tumour cell lines to accumulate isopentenyl pyrophosphate, thus rendering them more susceptible to Vδ2+ T cell cytotoxicity. However, little is known about whether ZA renders other, non-malignant cell types susceptible. In this study we focussed on macrophages (Mϕs), as these cells have been shown to take up ZA. We differentiated peripheral blood monocytes from healthy donors into Mϕs and then treated them with IFN-γ or IL-4 to generate M1 and M2 Mϕs, respectively. We characterised these Mϕs based on their phenotype and cytokine production and then tested whether ZA rendered them susceptible to Vδ2+ T cell cytotoxicity. Consistent with the literature, IFN-γ-treated Mϕs expressed higher levels of the M1 markers CD64 and IL-12p70, whereas IL-4-treated Mϕs expressed higher levels of the M2 markers CD206 and chemokine (C-C motif) ligand 18. When treated with ZA, both M1 and M2 Mϕs became susceptible to Vδ2+ T cell cytotoxicity. Vδ2+ T cells expressed perforin and degranulated in response to ZA-treated Mϕs as shown by mobilisation of CD107a and CD107b to the cell surface. Furthermore, cytotoxicity towards ZA-treated Mϕs was sensitive-at least in part-to the perforin inhibitor concanamycin A. These findings suggest that ZA can render M1 and M2 Mϕs susceptible to Vδ2+ T cell cytotoxicity in a perforin-dependent manner, which has important implications regarding the use of ZA in cancer immunotherapy.

Effect of the Modification of p24 Peptide Antigen on Dendritic Cell Uptake and T Cell Activation.

BACKGROUND: Vacc-4x is a candidate therapeutic vaccine consisting of 4 modified peptides based on conserved regions of HIV-1 p24Gag. Vacc4x has been shown to induce long term cellular immunity in immunized infected individuals resulting a reduction in viral load on treatment interruption. OBJECTIVE: Vacc-4x peptides are modified. In this study the effect of modification on uptake of the peptides into PBMC, their subsequent presentation and antigenicity was tested. The feasibility of using an in vitro culture system for testing immunogenicity of peptides using PBMC from uninfected donors was also assessed. METHODS: Labelled peptides were evaluated for uptake into PBMC using flow cytometry or confocal microscopy. Monocyte derived dendritic cells (DC) and autologous T cells were co-cultured with native and modified peptide antigens derived from p24. Activation was measured by flow cytometry and IFN-γ ELISPOT. RESULTS: Peptide modifications significantly increased peptide uptake by monocyte derived dendritic cells. Both the native (unmodified) and Vacc-4x (modified) peptide loaded DC could activate CD4+ and CD8+ T cell responses in vitro. Individual modified peptides induced greater responses than their native counterparts. The grouped Vacc-4x peptides elicited greater IFN γ responses than their native grouped counterparts at a lower concentration, however this effect was not detected at higher concentrations. CONCLUSION: These data indicate that the modifications increase uptake, alter the antigenicity of HIV- 1 p24 Vacc-4x peptides and increase the breadth of the response to the Vacc-4x peptides. The in vitro cell culture system is a suitable model for the antigenic assessment of peptide antigens.

Zoledronic acid causes γδ T cells to target monocytes and down-modulate inflammatory homing.

Zoledronic acid (ZA) is a potential immunotherapy for cancer because it can induce potent γδ T-cell-mediated anti-tumour responses. Clinical trials are testing the efficacy of intravenous ZA in cancer patients; however, the effects of systemic ZA on the activation and migration of peripheral γδ T cells remain poorly understood. We found that γδ T cells within ZA-treated peripheral blood mononuclear cells were degranulating, as shown by up-regulated expression of CD107a/b. Degranulation was monocyte dependent because CD107a/b expression was markedly reduced in the absence of CD14(+) cells. Consistent with monocyte-induced degranulation, we observed γδ T-cell-dependent induction of monocyte apoptosis, as shown by phosphatidylserine expression on monocytes and decreased percentages of monocytes in culture. Despite the prevailing paradigm that ZA promotes tumour homing in γδ T cells, we observed down-modulation of their tumour homing capacity, as shown by decreased expression of the inflammatory chemokine receptors CCR5 and CXCR3, and reduced migration towards the inflammatory chemokine CCL5. Taken together our data suggest that ZA causes γδ T cells to target monocytes and down-modulate the migratory programme required for inflammatory homing. This study provides novel insight into how γδ T cells interact with monocytes and the possible implications of systemic use of ZA in cancer.

Tripartite immune cell co-operation in the Bacillus Calmette Guérin-induced activation of γδ T cells.

γδ T cells contribute to immunosurveillance of pathogenic infections and malignant transformations; however, mechanisms of activation have yet to be fully defined. In this study we demonstrate a novel mechanism by which human Vδ2(+) γδ T cells are activated by the model pathogen Bacillus Calmette Guérin (BCG). We show in vitro that Vδ2 cell cytokine production and cytotoxic activity in response to BCG are dependent on both dendritic cells (DCs) and memory CD4(+) αß T cells (CD4 T cells). We found that Vδ2 cells are indirectly activated by BCG in an interleukin (IL)-12p70-dependent manner, and that DC production of the IL-12p70 responsible for Vδ2 cell activation requires Toll-like receptor 2/4 ligands from BCG and interferon (IFN)-γ from memory CD4 T cells. Our data suggest that Vδ2 cell responses to BCG are dependent on the activation of IFN-γ-producing memory CD4 T cells, and provide novel insight into the complex interplay between cells of the innate and adaptive immune response.

Mycobacteria activate γδ T-cell anti-tumour responses via cytokines from type 1 myeloid dendritic cells: a mechanism of action for cancer immunotherapy.

Attenuated and heat-killed mycobacteria display demonstrable activity against cancer in the clinic; however, the induced immune response is poorly characterised and potential biomarkers of response ill-defined. We investigated whether three mycobacterial preparations currently used in the clinic (BCG and heat-killed Mycobacterium vaccae and Mycobacterium obuense) can stimulate anti-tumour effector responses in human γδ T-cells. γδ T-cell responses were characterised by measuring cytokine production, expression of granzyme B and cytotoxicity against tumour target cells. Results show that γδ T-cells are activated by these mycobacterial preparations, as indicated by upregulation of activation marker expression and proliferation. Activated γδ T-cells display enhanced effector responses, as shown by upregulated granzyme B expression, production of the T(H)1 cytokines IFN-γ and TNF-α, and enhanced degranulation in response to susceptible and zoledronic acid-treated resistant tumour cells. Moreover, γδ T-cell activation is induced by IL-12, IL-1ß and TNF-α from circulating type 1 myeloid dendritic cells (DCs), but not from type 2 myeloid DCs or plasmacytoid DCs. Taken together, we show that BCG, M. vaccae and M. obuense induce γδ T-cell anti-tumour effector responses indirectly via a specific subset of circulating DCs and suggest a mechanism for the potential immunotherapeutic effects of BCG, M. vaccae and M. obuense in cancer.

Poly(I:C)-induced tumour cell death leads to DC maturation and Th1 activation.

Dendritic cells (DCs) have the ability to generate peptide epitopes for MHC class I molecules derived from apoptotic tumour cells for direct recognition by cytotoxic T cells. This function has lead to DCs being used in vaccine strategies. In this study, we investigate the effect of inducing apoptosis in tumour cell lines using IFN-γ and poly(I:C), the subsequent maturation of the endocytosing DC and its ability to direct the resulting T cell response. We show that uptake of poly(I:C)-induced apoptotic tumour cells leads to DC maturation and activation with a Th1 cell polarising capacity. In contrast, these effects are not seen by DCs loaded with γ-irradiated apoptotic tumour cells. We propose that the manner in which tumour cells are induced to die can have a profound effect on the endocytosing DC and the resulting T cell response.

Current status and future applications of cellular therapies for cancer.

Therapies based on the use of autologous immune cells are among the best candidates for cancer immunotherapy. Dendritic cell vaccines have demonstrated very encouraging responses for some solid tumors, while in melanoma autologous T-cell therapies have exceeded 70% objective response rates in selected Phase I trials. However, it is clear that a number of barriers exist to the effective, practical application of these therapies. The aim of this article is to consider modifications to such strategies over the last 3 years and the resultant clinical research in autologous dendritic cell vaccines, T-cell therapy and γδ T-cell therapy for cancer.

Impact of surgery on immunologic function: comparison between minimally invasive techniques and conventional laparotomy for surgical resection of colorectal tumors.

BACKGROUND: Surgical trauma suppresses host immune function, potentially creating an environment vulnerable to tumor cell growth. This study compared immune function after laparoscopy, minilaparotomy, and conventional colorectal tumor resections. METHODS: Seventy-one patients underwent surgery (20 laparoscopy, 21 minilaparotomy, and 30 conventional). Blood samples were taken before surgery and at 3 hours, 24 hours, and 5 days after surgery. White blood cell constitution was determined using monoclonal antibodies. Levels of TH1 cytokines interferon-gamma, tumor necrosis factor-alpha, and interleukin (IL)-2 and TH2 cytokines IL-10, -4, and -6 were measured in plasma and from supernatants of activated peripheral blood mononuclear cells. RESULTS: At 5 days after surgery, lymphocyte counts remained low in the conventional and minilaparotomy groups (P = .001 and P = .008) but had resolved in laparoscopic patients. Three-hour postoperative serum IL-6 concentrations were lower in laparoscopic than in conventional patients (P = .028). Production of TH1 cytokines 3 hours after surgery were significantly increased in laparoscopic patients (interferon-gamma P = .018, tumor necrosis factor-alpha P = .011, and IL-2 P = .037). CONCLUSIONS: TH1 lymphocyte function is improved transiently and immune homeostasis restored earlier in patients undergoing laparoscopic colorectal cancer resection, which may influence disease recurrence.

Treatment of murine collagen-induced arthritis by the stress protein BiP via interleukin-4-producing regulatory T cells: a novel function for an ancient protein.

OBJECTIVE: Following the demonstration that the stress protein, BiP, prevented induction of collagen-induced arthritis (CIA) in HLA-DRB*0101+/+ (HLA-DR1+/+) mice, we investigated the immunotherapeutic ability of BiP to suppress disease during the active phase of CIA in HLA-DR1+/+ and DBA/1 mice. METHODS: BiP was administered either subcutaneously or intravenously to DBA/1, HLA-DR1+/+, or interleukin-4 (IL-4)-knockout mice at the onset of arthritis. Immune cells were used in adoptive transfer studies or were restimulated in culture with BiP or type II collagen (CII). Proliferation and cytokine release were measured. In addition, serum anti-CII antibodies were measured by enzyme-linked immunosorbent assay. Disease progression was scored using a visual analog scale. RESULTS: BiP was successful in suppressing established CIA in HLA-DR1+/+ and DBA/1 mice. Serum levels of anticollagen IgG antibodies were reduced in BiP-treated mice. T cells from BiP-immunized mice produced Th2 cytokines, in particular, IL-4. Treatment with BiP was also shown to increase the production of CII-specific IL-5, IL-10, and interferon-gamma at the termination of the study. Development of severe CIA was prevented by the intravenous transfer of BiP-specific cells at the time of CIA induction in HLA-DR1+/+ mice or by transferring BiP-specific cells to DBA/1 mice at the onset of disease. BiP failed to ameliorate the development of CIA in IL-4-/-, HLA-DR1+/+ mice. CONCLUSION: These novel results show that BiP can suppress active CIA by the induction of regulatory cells that act predominantly via IL-4. Thus, BiP is a potential immunotherapeutic agent for the treatment of patients with rheumatoid arthritis.

Inhibition of antigen-presenting cell function and stimulation of human peripheral blood mononuclear cells to express an antiinflammatory cytokine profile by the stress protein BiP: relevance to the treatment of inflammatory arthritis.

OBJECTIVE: The stress protein and endoplasmic reticulum chaperone, immunoglobulin binding protein (BiP), is an autoantigen in rheumatoid arthritis (RA). Stress proteins, however, may have extracellular functions, mediated via cell surface receptors, that may include immunomodulatory functions. We sought to determine whether cell-free BiP is present in the synovial fluid (SF) of patients with RA and to further investigate the possible extracellular antiinflammatory and immunomodulatory properties of BiP in peripheral blood mononuclear cells (PBMCs) in vitro. METHODS: The presence of BiP in SF was established by Western blotting. PBMCs were stimulated with exogenous recombinant human BiP, and cytokine production and cell proliferation were measured in the presence and absence of cell signaling inhibitors or neutralizing anti-interleukin-10 (anti-IL-10) monoclonal antibody. Cytokine levels were quantified by enzyme-linked immunosorbent assay, cell proliferation by tritiated thymidine uptake, and cell surface molecule expression by flow cytometry. RESULTS: PBMCs responded to BiP with secretion of an antiinflammatory profile of cytokines. Although BiP stimulated the early production of tumor necrosis factor alpha (TNF alpha), the major cytokine induced was IL-10. Soluble TNF receptor II and IL-1 receptor antagonist secretion was also increased. Addition of SB203580, the MAPK p38 pathway inhibitor, partially inhibited the production of IL-10 and TNF alpha, whereas they were unaffected by the MAPK ERK-1/2 inhibitor PD98059. BiP also inhibited the recall antigen response by PBMCs to tuberculin purified protein derivative. Further investigation showed that incubation of monocytes in the presence of either BiP or IL-10 down-regulated CD86 and HLA-DR expression. The effect observed with IL-10 was transient compared with the long-lasting reduction induced by BiP. CONCLUSION: Extracellular BiP may stimulate immunomodulatory and antiinflammatory pathways, which are only partly due to the production of IL-10. These properties may be of relevance for the treatment of diseases such as RA.

Serologic changes following B lymphocyte depletion therapy for rheumatoid arthritis.

OBJECTIVE: To explore the changes in serologic variables and clinical disease activity following B lymphocyte depletion in 22 patients with rheumatoid arthritis (RA). METHODS: B lymphocyte depletion was attained using combination therapy based on the monoclonal anti-CD20 antibody rituximab. Levels of a serologic indicator of inflammation, C-reactive protein (CRP), of antimicrobial antibodies, of autoantibodies including IgA-, IgM-, and IgG-class rheumatoid factors (RF), and of antibodies to cyclic citrullinated peptide (anti-CCP) were assayed. RESULTS: The majority of patients showed a marked clinical improvement after treatment with rituximab, with benefit lasting up to 33 months. Levels of total serum immunoglobulins fell, although the mean values each remained within the normal range. Whereas the IgM-RF response paralleled the changes in total serum IgM levels, the levels of IgA-RF, IgG-RF, and IgG and anti-CCP antibodies decreased significantly more than did those of their corresponding total serum immunoglobulin classes. The kinetics for the reduction in CRP levels also paralleled the decreases in autoantibody levels. In contrast, levels of antimicrobial antibodies did not change significantly. B lymphocyte return occurred up to 21 months posttreatment. The time to relapse after B lymphocyte return was often long and unpredictable (range 0-17 months). Relapse was, however, closely correlated with rises in the level of at least one autoantibody. Increased autoantibody levels were rarely observed in the absence of clinical change. CONCLUSION: Following B lymphocyte depletion in patients with RA, a positive clinical response occurred in correlation with a significant drop in the levels of CRP and autoantibodies. Antibacterial antibody levels were relatively well maintained. B lymphocyte return preceded relapse in all patients. There was also a temporal relationship between clinical relapse and rises in autoantibody levels. Although these observations are consistent with a role for B lymphocytes in the pathogenesis of RA, the precise mechanisms involved remain unclear.